Date of Award
Doctor of Philosophy
Plants, Soils, and Insects
C. Neal Stewart
Nicole Labbe, Feng Chen, Jay Chen
The natural recalcitrance of plant cell walls is a major commercial hurdle for plant biomass to be converted into a viable energy source as alternative to fossil fuels. To circumvent this hurdle manipulation of carbohydrate enzymes active in the cellulose and hemicellulose portions of the plant cell wall can be utilized to improve feedstocks. Production of cellulolytic enzymes by plants have been evaluated for reducing the cost associated with lignocellulosic biofuels. Plants have successfully served as bioreactors producing bacterial and fungal glycosyl hydrolases, which have altered plant growth to improve saccharification. A bioprospecting opportunity lies with the utilization of insect glycosyl hydrolases for transgenic production in plants. Lessons learned from microbial hydrolase expression can be applied to insect hydrolase expression along with gene stacking to develop autohydrolysis plant lines.
A step toward production of insect cellulases in plants was performed by insertion of the endo-glucanase TcEG1 gene, from Tribolium castaneum, into switchgrass. Transgenic lines overexpressing TcEG1 produced a functional enzyme with an optimal alkaline pH activity of 12.0. Recalcitrance was assayed by performing saccharification analysis, in which one line was superior over non-transgenic control; this line also had reduced 9% lignin content. Transgenic lines developed narrow stems, although biomass yield was unchanged due to increased tiller number and cell wall thickness.
Grasses contain a relatively high amount of glucoarabinoxylan in their cell walls, which cross links with lignin. By down-regulation of a uridine diphosphate arabinomutase (UAM) gene via RNAi, it was hypothesized that attenuated production of this carbohydrate transferase would increase saccharification of switchgrass biomass from a disruption of cross linking. Transgenic events showed a reduction in arabinose content (up to 58%) and altered arabino-side chains, however saccharification was unchanged. UAM transgenic switchgrass showed a red node phenotype, which could be in response to increased lignin biosynthesis. A model of UAM-influenced cell wall interactions was proposed and will used to build hypotheses for future-omics research.
In summary, switchgrass saccharification and biomass yield can be increased by introduction of carbohydrate active enzymes. Combination of presented transgenic lines with low-lignin germplasm could be utilized to further improve saccharification yield.
Willis, Jonathan Duran, "Modification of carbohydrate active enzymes in switchgrass (Panicum virgatum L.) to improve saccharification and biomass yields for biofuels. " PhD diss., University of Tennessee, 2016.